1. Field of the Invention
The present invention relates to linked fusion polypeptides derived from single and multiple chain proteins. In particular, the invention relates to the linker peptide essential for bridging the polypeptide constituents that comprise the linked fusion polypeptide.
2. Description of the Background Art
The advent of modern molecular biology and immunology has brought about the possibility of producing large quantities of biologically active materials in highly reproducible form and with low cost. Briefly, the gene sequence coding for a desired natural protein is isolated, replicated (cloned) and introduced into a foreign host such as a bacterium, a yeast (or other fungi) or a mammalian cell line in culture, with appropriate regulatory control signals. When the signals are activated, the gene is transcribed and translated, and expresses the desired protein. In this manner, such useful biologically active materials as hormones, enzymes and antibodies have been cloned and expressed in foreign hosts.
One of the problems with this approach is that it is limited by the "one gene, one polypeptide chain" principle of molecular biology. In other words, a genetic sequence codes for a single polypeptide chain. Many biologically active polypeptides, however, are aggregates of two or more chains. For example, antibodies are three-dimensional aggregates of two heavy and two light chains. In the same manner, large enzymes such as aspartate transcarbamylase, for example, are aggregates of six catalytic and six regulatory chains, these chains being different. In order to produce such complex materials by recombinant DNA technology in foreign hosts, it becomes necessary to clone and express a gene coding for each one of the different kinds of polypeptide chains. These genes can be expressed in separate hosts. The resulting polypeptide chains from each host would then have to be reaggregated and allowed to refold together in solution. Alternatively, the two or more genes coding for the two or more polypeptide chains of the aggregate could be expressed in the same host simultaneously, so that refolding and reassociation into the native structure with biological activity will occur after expression. This approach, however, necessitates expression of multiple genes in a single host.
A classical example of multigene expression to form multimeric polypeptides is the expression by recombinant DNA technology of antibodies. Antibodies are immunoglobulins typically composed of four polypeptides; two heavy chains and two light chains. Genes for heavy and light chains have been introduced into appropriate hosts and expressed, followed by reaggregation of these individual chains into functional antibody molecules (see, for example, Munro, Nature 312:597 (1984); Morrison, S. L., Science 229:1202' (1985); and Oi et al., BioTechniques 4:214 (1986); Wood et al., Nature 314:446-449 (1985)).
Antibody molecules have two generally recognized regions in each of the heavy and light chains. These regions are the so-called "variable" region which is responsible for binding to the specific antigen in question, and the so-called "constant" region which is responsible for biological effector responses such as complement binding, etc. The constant regions are not necessary for antigen binding. The constant regions have been separated from the antibody molecule, and biologically active (i.e., binding) variable regions have been obtained.
The variable regions of a light chain (V.sub.L) and a heavy chain (V.sub.H) together form the structure responsible for an antibody's binding capability. Light and heavy chain variable regions have been cloned and expressed in foreign hosts, and maintain their binding ability (Moore et al., European Patent Publication 0088994 (published Sep. 21, 1983) see also Cabilly, U.S. Pat. No. 4,816,567 (issued Mar. 28, 1989)). Antibodies may be cleaved to form fragments, some of which retain their binding ability. One such fragment is the "Fv" fragment, which is composed of the terminal binding portions of the antibodies. The Fv comprises two complementary subunits, the V.sub.L and V.sub.H, which in the native antibody compose the binding domains.
The Fv fragment of an antibody is probably the minimal structural component which retains the binding characteristics of the parent antibody. The limited stability at low protein concentrations of the Fv fragments may be overcome by using an artificial peptide linker to join the variable domains of an Fv. The resulting single-chain Fv (hereinafter "sFv") polypeptides have been shown to have binding affinities equivalent to the monoclonal antibodies (MAbs) from which they were derived (Bird et al., Science 242:423 (1988)). In addition, catalytic MAbs may be converted to a sFv form with retention of catalytic characteristics (Gibbs et al., Proc. Natl. Acad. Sci., USA 88:4001 (1991)).
There are a number of differences between single-chain Fv (sFv) polypeptides and whole antibodies or antibody fragments, such as Fab or F(ab)'.sub.2. Single-chain Fv polypeptides are small proteins with a molecular weight around 27 kd, which lack the constant regions of 50 kd Fab fragments or 150 kd immunoglobulin antibodies bearing gamma chains (IgG). Like a Fab fragment, and unlike an IgG, an sFv polypeptide contains a single binding site.
The in vivo properties of sFv polypeptides are different from MAbs and antibody fragments. Due to their small size, sFv polypeptides clear more rapidly from the blood and penetrate more rapidly into tissues (Colcher, et al., J. Natl. Cancer Inst. 82:1191 (1990); Yokota et al., Cancer Research 52:3402 (1992)). Due to lack of constant regions, sFv polypeptides are not retained in tissues such as the liver and kidneys. Due to the rapid clearance and lack of constant regions, sFv polypeptides will have low immunogenicity. Thus, sFv polypeptides have applications in cancer diagnosis and therapy, where rapid tissue penetration and clearance are advantageous.
Monoclonal antibodies have long been envisioned as magic bullets, in which they deliver to a specific tumor cell a cytotoxic agent in a highly targeted manner. sFv polypeptides can be engineered with the two variable regions derived from a MAb. The sFv is formed by ligating the component variable domain genes with an oligonucleotide that encodes an appropriately designed linker polypeptide. Typically, the linker bridges the C-terminus of the first V region and the N-terminus of the second V region. sFv polypeptides offer a clear advantage over MAbs because they do not have the constant regions derived from their biological source, which may cause antigenic reaction against the MAb. Single-chain immunotoxins have been produced by fusing a cell binding sFv with Pseudomonas exotoxin (Chaudhary et al., Nature 339:394 (1989)). Recently, a single-chain immunotoxin was shown to cause tumor regression in mice (Brinkmann et al., Proc. Natl. Acad. Sci. USA 88:8616 (1991)).
The general considerations behind the design and construction of polypeptide linkers as applied to sFv polypeptides have been previously described in U.S. Pat. No. 4,946,778 (Ladner et al.). Computer design of linkers has also been described in U.S. Pat. Nos. 4,704,692, 4,853,871, 4,908,773 and 4,936,666.
Four linkers are described in the '778 disclosure: TRY40, TRY 59, TRY61, and TRY104b. TRY40 is a double linker with 3- and 7-amino acid sequences comprising the linkers. The sequences are PGS and IAKAFKN (see page 8, Table 1 for a description of the single letter amino acid code used herein). TRY59 is an 18-residue single linker having the sequence KESGSVSSEQLAQFRSLD (SEQ. ID No. 2). TRY 61 is a 14-residue single linker having the sequence VRGSPAINVAVHVF (SEQ. ID No. 3). TRY104b is a 22-residue single linker constructed primarily of a helical segment from human hemoglobin. The sequence is AQGTLSPADKTNV KAAWGKVMT (SEQ. ID No. 4).
Traunecker et al., EMBO J. 10(12):3655-3659 (1991) have disclosed an 18-amino acid linker for joining the first two N-terminal CD4 domains and the combining site of the human CD3 complex. Its sequence is VEGGSGGS GGSGGSGGVD (SEQ. ID No. 5). The final bispecific single-chain polypeptide is called Janusin, and targets cytotoxic lymphocytes on HIV-infected cells.
Fuchs et al., Bio/Technology 9:1369-1372 (1991), used an 18-residue linker to join the heavy- and light-chain variable domains of a humanized antibody against chick lysozyme. The 18-residue linker was partially derived from .alpha.-tubulin and contains a MAb epitope specific to .alpha.-tubulin. The full sequence is GSASAPKLEEGEFSEARE (SEQ. ID No. 6).
A host of single-chain Fv analog polypeptides are disclosed in the literature (see, Huston, J. S. et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Huston, J. S. et al., SIM News 38(4) (Suppl.):11 (1988); McCartney, J. et al., ICSU Short Reports 10:114 (1990); McCartney, J. E. et al., unpublished results (1990); Nedelman, M. A. et al., J. Nuclear Med. 32 (Suppl.):1005 (1991); Huston, J. S. et al., In: Molecular Design and Modeling: Concepts and Applications, Part B, edited by J. J. Langone, Methods in Enzymology 203:46-88.(1991); Huston, J. S. et al., In: Advances in the Applications of Monoclonal Antibodies in Clinical Oncology, Epenetos, A. A. (Ed.), London, Chapman & Hall (1993); Bird, R. E. et al., Science 242:423-426 (1988); Bedzyk, W. D. et al., J. Biol. Chem. 265:18615-18620 (1990); Colcher, D. et al., J. Nat. Cancer Inst. 82:1191-1197 (1990); Gibbs, R. A. et al., Proc. Natl. Acad. Sci. USA 88:4001-4004 (1991); Milenic, D. E. et al., Cancer Research 51:6363-6371 (1991); Pantoliano, M. W. et al., Biochemistry 30:10117-10125 (1991); Chaudhary, V. K. et al., Nature 339:394-397 (1989); Chaudhary, V. K. et al., Proc. Natl. Acad. Sci. USA 87:1066-1070 (1990); Batra, J. K. et al., Biochem. Biophys. Res. Comm. 171:1-6 (1990); Batra, J. K. et al., J. Biol. Chem. 265:15198-15202 (1990); Chaudhary, V. K. et al., Proc. Natl. Acad. Sci. USA 87:9491-9494 (1990); Batra, J. K. et al., Mol. Cell. Biol. 11:2200-2205 (1991); Brinkmann, U. et al., Proc. Natl. Acad. Sci. USA 88:8616-8620 (1991); Seetharam, S. et al., J. Biol. Chem. 266:17376-17381 (1991); Brinkmann, U. et al., Proc. Natl. Acad. Sci. USA 89:3075-3079 (1992); Glockshuber, R. et al., Biochemistry 29:1362-1367 (1990); Skerra, A. et al., Bio/Technol. 9:273-278 (1991); Pack, P. et al., Biochem. 31:1579-1534 (1992); Clackson, T. et al., Nature 352:624-628 (1991); Clackson, T. et al., Nature 352:624-628 (1991); Marks, J. D. et al., J. Mol. Biol. 222:581-597 (1991); Iverson, B. L. et al., Science 249:659-662 (1990); Roberts, V. A. et al., Proc. Natl. Acad. Sci. USA 87:6654-6658 (1990); Condra, J. H. et al., J. Biol. Chem. 265:2292-2295 (1990); Laroche, Y. et al., J. Biol. Chem. 266:16343-16349 (1991); Holvoet, P. et al., J. Biol. Chem. 266:19717-19724 (1991); Anand, N. N. et al., J. Biol. Chem. 266:21874-21879 (1991); Fuchs, P. et al., Bio/Technol. 9:1369-1372 (1991); Breitling, F. et al., Gene 104:104-153 (1991); Seehaus, T. et al., Gene 114: in press (1992); Takkinen, K. et al., Prot. Eng. 4:837-841 (1991); Dreher, M. L. et al., J. Immunol. Methods 139:197-205 (1991); Mottez, E. et al., Eur. J. Imunol. 21:467-471 (1991); Traunecker, A. et al., Proc. Natl. Acad. Sci. USA 88:8646-8650 (1991); Traunecker, A. et al., EMBO J. 10:3655-3659 (1991); Hoo, W. F. S. et al., Proc. Natl. Acad. Sci. USA 89:4759-4763 (1993)). Linker lengths used in those Fv analog polypeptides vary from 10 to 28 residues.
Linkers previously used for sFvs and other polypeptides suffer from proteolytic attack, rendering them less stable and prone to dissociation. They also suffer from inordinate aggregation at high concentrations, making them susceptible to concentration in the liver and kidneys. Therefore, there is a need for more stable linkers that are resistant to proteolytic attack and less prone to aggregation.